Manipulation of gene copy number in bordetella

ABSTRACT

Protein expression levels from Bordetella strains, particularly Bordetella pertussis, are altered by genetic modification to a natural Bordetella strain whereby one or more of the natural genes, particularly including the TOX, FHA, CYA and PRN genes, is deleted from the genome of the natural strain and one or more of the natural genes or a genetic mutation thereof, particularly a genetically-detoxified TOX* gene, or a hybrid gene, is inserted into the genome of the natural strain to provide at least two copies of one or more of the natural genes or genetic mutation thereof or hybrid gene, singly or in tandem. The altered genotype Bordetella strain is useful in producing whole-cell or defined component vaccines against Bordetella, particularly whooping cough, which may be employed in combination with other vaccines.

FIELD OF INVENTION

The present invention relates to a novel approach to alter geneexpression in Bordetella species by manipulation of gene copy number.

BACKGROUND OF THE INVENTION

Whooping cough in humans is caused by members of the Bordetella species,especially B. pertussis. Following disease or vaccination, antibodiesare elicited against several bacterial proteins, especially pertussistoxin (PT), filamentous haemagglutinin (FHA), the 69kDa outer membraneprotein (69kD or pertactin) and the fimbrial agglutinogens. PT is amajor protective antigen and is also associated with virulence. Theseantigens have been proposed to formulate single or multi-componentpertussis vaccines and, as such, their efficient production andpurification are essential.

During fermentation of B. pertussis, it has been found that FHA issecreted at 7 and 10 times the molar levels of 69kD or PT, respectively.Therefore, PT and pertactin are limiting antigens in the production of acomponent vaccine. Furthermore, the relative overproduction of FHA makesthe purification of other antigens expressed at lower levels moredifficult. This is especially true for PT, which may be initiallyco-purified with FHA in some purification protocols. It is also ofconcern that active PT may contaminate the FHA preparation, which thenneeds to be chemically detoxified to inactivate the residual nativetoxin. In the case of genetically detoxified pertussis toxin (describedin published EPO patent application No. 0322115 and corresponding U.S.Pat. No. 5,085,862, assigned to the assignee hereof and the disclosureof which is incorporated herein by reference; Loosmore et al., Infect.Immun. 58, 3653 [1990]), this would not be a problem. In either case, itwould be advantageous, from a vaccine production viewpoint, to have a B.pertussis strain which does not produce FHA and/or one which does notproduce PT. For example, an FHA⁻ strain in which the FHA gene has beendeleted could be used to produce all other antigens under optimizedfermentation and purification conditions. Similarly, a PT⁻ strain wouldproduce FHA with no possibility of contamination by pertussis toxin.

There are B. pertussis gene products which are not necessary in awhooping cough vaccine, for example, dermonecrotic toxin (DNT) andpossibly adenylate cyclase toxin. The gene loci coding for such productscould be considered to be dispensable in a vaccine strain as long as thestrain remains viable and produces the other antigens in satisfactoryamounts. The deletion of dispensable genes offers the possibility offurther genetic manipulation by in situ gene insertion at the loci ofgene deletions. For example, the adenylate cyclase gene (CYA) could bedeleted and one or more additional copies of one of the candidatevaccine antigen genes inserted at the CYA locus. The additional gene(s)may be introduced through homologous recombination using the flankingsequences of the deleted gene to direct in situ gene insertion.Alternatively, gene copies also may be introduced through randominsertion into the chromosome or through the introduction of replicatingplasmids. Multiple copies of regulatory genes, such as the vir regulon,Bvg, could also be introduced to modify antigen expression.

Examples of deleted strains could be TOX⁻, FHA⁻, PRN⁻, CYA⁻, DNT⁻ or anycombination of these or other gene deletions. Inserted genes could beany of the TOX, FHA, PRN or any other native, mutated, or hybrid genesand one or more copies could be inserted in tandem or separately. Suchgenetic manipulations aimed at augmenting a gene copy number with orwithout deletion of other genes, could lead to substantial enhancementof Bordetella antigen production and/or optimization of antigenpurification.

Lee et al (1989, Infect. Immun. 57: 1413-1418) attempted to generatehypertoxigenic strains of B. pertussis by expression of the TOX operonfrom a multicopy replicating plasmid. No increase in PT production wasobserved, however, and the plasmid was rearranged, the TOX operondeleted or the transconjugants underwent conversion to an avirulentphase.

SUMMARY OF THE INVENTION

In accordance with the present invention, there is provided a novelmethod to alter gene expression in Bordetella species by themanipulation of gene copy number. One or more genes may be deleted fromany Bordetella strain and additional gene copies inserted singly or intandem to change protein expression levels.

Accordingly, in one aspect of the present invention, there is provided agenetically-modified Bordetella strain, particularly agenetically-modified Bordetella pertussis strain, having one or more ofthe natural genes deleted from the genome and one or more of the naturalgenes or a genetic mutation thereof or a hybrid gene, inserted into thegenome to provide at least two copies of one or more of the naturalgenes or genetic mutation thereof, or hybrid gene, in the genome, singlyor in tandem, to effect alteration of expression levels of proteinsencoded by genes present in the genetically-modified strain incomparison to the natural unmodified strains.

The altered expression level of the protein can lead to enhancement ofantigen production and optimization of purification of protein.

The altered genotype strains are useful in producing whole-cell or acellular defined component vaccines against Bordetella, particularlywhooping cough, which may be employed in combination with othervaccines.

BRIEF DESCRIPTION OF DRAWINGS

FIGS. 1A-1E

FIG. 1A shows the plasmids used to derive the copy number-alteredBordetella strains described herein. FIGS. 1B, 1C, 1D and 1E describetheir construction. pGZ71 contains an E. coli S12 and tetracyclineresistance gene cassette, as described in U.S. Pat. No. 5,085,862,sandwiched between the FHA gene flanking regions. Plasmid S-3741-2 (ATCC75031) contains a single copy of TOX sandwiched between the 5' and 3'FHA gene flanking regions. Plasmid S-3822-10 has tandem copies of themutant TOX operon (TOX*), which encodes the Lys9Gly129 PT analogue,sandwiched between the FHA flanking regions. Plasmids S-3980-9 andS-4000-5 contain tandem copies of TOX or TOX*, respectively, between the5' and 3' TOX flanking regions. Plasmid pRY46-10 has the S12/Tet^(R)cassette between the 5' and 3' PRN flanking regions. To increase theproduction of pertactin, the natural promoter was replaced by the FHApromoter to produce a hybrid gene FHApPRN, as described in publishedEuropean patent application No. EP 453,216, in the name of the assigneehereof. Plasmid S-4258-7 contains two tandem copies of a hybrid FHApPRNgene.

FIG. 1B illustrates the construction of plasmids pGZ71 and S-3741-2which is further described in Examples 4 and 5. FIG. 1C illustrates theconstruction of plasmids S-3822-10 and S-4000-5, further described inExamples 6 and 7. FIG. 1D illustrates the construction of plasmidpRY46-10, which is described in Example 10. FIG. 1E illustrates theconstruction of plasmid S-4258-7, which is described in Example 11.Restriction sites are B, BamH I; Bal, Bal I; Bg, Bgl II; C, Cla I; H,Hind III; K, Kpn I; Nco, NcoI; R, EcoR I; S, Sal I; Sm, Sma I; and Su,Sau3A I.

FIG. 2A shows a schematic representation of the genes present at the TOXand FHA loci and FIG. 2B shows the Southern blot analysis for B.pertussis strains 10536, 989-56, 191-134, and 890-393. Lane 1, strain10536, the parental wild-type (wt) strain; lane 2, strain 989-56containing one copy of TOX*; lane 3, strain 191-134 containing tandemcopies of TOX* at the TOX locus; and lane 4, strain 890-393 containingthree copies of TOX* one at the TOX locus and tandem copies at the FHAlocus. DNA was digested with Bgl II and Cla I and probed with aTOX-specific DNA fragment representing the entire TOX operon.Integration of additional copies of the TOX operon is shown by theappearance of new TOX-specific hybridization fragments superimposed uponthe wild-type pattern. Restriction sites are Bg, Bgl II; C, Cla I; andR, EcoR I.

FIG. 3 shows the kinetics of PT or PT analogue expression for strains10536 (wt), and 890-393 (3 TOX*) when grown in 10 liter bioreactors.

FIGS. 4A and 4C shows a comparative analysis by reverse phase HPLC andFIG. 4B shows a comparative analysis by SDS-PAGE of pertussis toxinanalogue (S1 Lys9Gly129) purified from culture supernatants of strains989-56 (TOX*) and 890-393 (3 TOX*), indicating that they arestructurally indistinguishable.

FIGS. 5A-5B

FIG. 5A demonstrates the effect of heptakis(2,6-O-dimethyl)β-cyclodextrin on the expression of PT from strain10536, when grown in a 10 liter fermentor. FIG. 5B demonstrates theeffect of β-cyclodextrin on strain 1190-74 (2 TOX) when grown in a 10liter fermentor.

FIG. 6A shows a schematic representation of the PRN locus and FHApPRNhybrid genes and FIG. 6B shows the Southern blot analysis for B.pertussis strains 10536, 1090-108-3, 591-473, and 1091-297. Lanes 1 and3, strain 10536, the wild-type parent digested with Dde I and Sal I,respectively; lanes 2 and 5, strain 591-473, containing one copy of theFHApPRN gene, digested with Dde I and Sal I, respectively; lane 4,strain 1090-108-3, the PRN deleted strain containing the S12/Tet^(R)cassette, digested with Sal I; lane 6, strain 1091-297 containing twocopies of the FHApPRN hybrid gene, digested with Sal I. The restrictedDNA was probed with a PRN-specific DNA fragment representing the entirePRN gene. Integration of tandem copies of the FHApPRN hybrid gene isshown by the appearance of new PRN-specific hybridization fragmentssuperimposed upon the wild-type pattern. Restriction sites are A, Apa I;C, Cla I; D, Dde I; S, Sal I.

FIGS. 7A-7B

FIG. 7A shows the kinetics of antigen expression from B. pertussisstrain 1091-107 when grown in 10 liter fermentors. Strain 1091-107contains two copies of the wild-type TOX operon integrated at the TOXlocus and a single copy of the FHApPRN hybrid gene integrated at the PRNlocus. FIG. 7B shows the kinetics of antigen production by B. pertussisstrain 1091-359 when grown in 10 liter fermentors. Strain 1091-359contains two copies of the TOX* operon, encoding the Lys9Gly129 PTanalogue, at the TOX locus and a single copy of the FHApPRN hybrid geneintegrated at the PRN locus.

DEPOSIT INFORMATION

Certain biological materials described herein for the first time havebeen deposited with the American Type Culture Collection (ATCC) locatedat Rockville, Md., U.S.A. pursuant to the Budapest Treaty and prior tothe filing of this application, identified as follows:

    ______________________________________                                        (a) Bordetella strains:                                                       Strain No.   Accession No. Deposit Date                                       ______________________________________                                        191-134      55,205        June 18, 1991                                      890-393      55,206        June 18, 1991                                      1091-107     55,313        April 2, 1992                                      1091-359     55,312        April 2, 1992                                      591-473      55,321        April 30, 1992                                     1091-27      55,462        August 17, 1993                                    ______________________________________                                        (b) Plasmid DNA:                                                              Plasmid No.  Accession No. Deposit Date                                       ______________________________________                                        S-3741-2     75031         June 18, 1991                                      S-3980-9     75033         June 18, 1991                                      ______________________________________                                    

Other deposited biological materials also are referred to and identifiedherein but have been deposited in connection with other patent andnon-patent activities. Cultures of the deposited microorganisms andplasmids will become available to the public on the grant of a patent onthis United States patent application. The invention described andclaimed herein is not to be limited in scope by the strains ofmicroorganisms and plasmids deposited, since the deposited embodiment isintended only as an illustration of the invention. Any equivalentmicroorganisms or plasmids that produce equivalent changes in antigenproduction as described in this application are within the scope of theinvention.

GENERAL DESCRIPTION OF INVENTION

The genes for PT (TOX), FHA (FHA), pertactin (PRN), and adenylatecyclase (CYA) have been cloned and sequenced (Nicosia et al., Proc.Natl. Acad. Sci. U.S.A. 83, 4631 [1986]; Loosmore et al., Nucl. AcidsRes. 17, 8365 [1989]; Relman et al., Proc. Natl. Acad. Sci. U.S.A. 86,2637 [1989]; Charles et al., Proc. Natl. Acad. Sci. U.S.A. 86, 3554[1989]; Glaser et al., Molec. Microbiol. 2, 19 [1988]). Pertussis toxinis a complex protein composed of six polypeptide subunits, encoded byfive different structural genes expressed from a single promoter. Itsenzymatic and most of its toxic activities are mediated by its subunitS1, while its cell-binding and mitogenic properties are due to the othersubunits, which form the B-subunit. Site-directed mutagenesis ofselected codons of the S1 and/or B subunit genes leads to detoxificationof the holotoxin (EPO 322115, U.S. Pat. No. 5,085,862, and Loosmore etal., Infect. Immun. 58 3653 [1990]).

Deletion of the TOX operon by homologous recombination followingelectroporetic transformation of DNA into Bordetella pertussis has beendescribed in EPO 322,115 and U.S. Pat. No. 5,085,862. Briefly, thedeletion of a gene in a streptomycin-resistant B. pertussis strain usingan S12/Tet^(R) gene cassette, produces a streptomycin sensitive,tetracycline resistant phenotype. Replacement of the selectablecartridge by a further round of recombination results in streptomycinresistance. This procedure allows specific gene replacement in B.pertussis without the integration of an antibiotic resistance gene. TheS12/Tet^(R) gene cassette was introduced into the TOX locus by allelicexchange using a 2.9 kb Sau3A I/EcoR I fragment of TOX 5'-flankingregion and a 5 kb EcoR I/Sal I fragment of TOX 3'-flanking region. Thereplacement of the gene cassette by in vitro mutated alleles has beendescribed (EPO 322,115, U.S. Pat. No. 5,085,862; Pizza et al., Science246, 497 [1989]; Loosmore et al., Infect. Immun. 58, 3653 [1990]; Zealeyet al., Bio/Technology 8, 1025 [1990]). Deletion of the FHA and PRNgenes was performed using a similar technique. These techniques are usedherein to engineer B. pertussis strains having duplicated genes at theiroriginal and/or new and novel genetic loci (Tables 1A and 1B). Theseexperiments were approved by the Connaught Biosafety Committee and wereperformed under either level 2 or 3 containment.

B. pertussis strain 29-8 (ATCC 53973) is a derivative of the Connaughtvaccine strain 10536 in which the TOX operon has been deleted andreplaced by a cassette containing the E. coli S12 and tetracyclineresistance genes (Zealey et al., Bio/Technology 8, 1025 [1990]; EPO322,115, U.S. Pat. No. 5,085,862). Through homologous recombination, theS12/Tet^(R) gene cassette is replaced by tandem copies of TOX togenerate a strain (1190-74) which produces approximately two to threetimes the amount of PT produced by the wild-type strain. Similarly,tandem TOX* operons mutated to engineer a genetically detoxified PTanalogue having the mutations Arg9→Lys9 and Gly129→Glu129 in the S1subunit (Loosmore et al., Infect. Immun. 58, 3653 [1990]) have beenintroduced into strain 29-8 to generate strain 191- 134 (ATCC 55205).

Strain 390-101 (ATCC 55157) is a derivative of the Connaught vaccinestrain 10536 in which the FHA gene locus has been replaced by theS12/Tet^(R) gene cassette. An additional TOX operon has been inserted atthe FHA locus of this strain to generate strain 590-208, which producesapproximately twice as much PT as strain 10536, but no FHA.

Strain 989-56 (ATCC 53975) is a derivative of strain 10536 whichexpresses the PT analogue with Lys9Gly129 mutations in S1 and isdescribed in copending U.S. patent application Ser. No. 589,423 filedSep. 18, 1990, assigned to the assignee hereof, the disclosure of whichis incorporated herein by reference. Replacement of its FHA gene by theS12/Tet^(R) gene cassette led to the TOX*/FHA⁻ strain 490-324. Twoadditional tandem TOX* operons have been introduced at the FHA locus togenerate a strain with three copies of TOX*, strain 890-393 (ATCC55206), which expresses approximately three times the amount of PTanalogue.

Using the same techniques, the PRN gene was deleted from strain 10536 togive strain 1090-108-3 (ATCC 55156). The PRN locus can similarly be usedto introduce multiple copies of PRN or hybrid operons. The engineeringof hybrid operons is described in our published European patentapplication EPO 453,216 and corresponding U.S. patent application Ser.No. 687,231 filed Apr. 18, 1991, assigned to the assignee hereof and thedisclosure of which is incorporated herein by reference. Briefly, ahybrid operon consists of a structural gene from Bordetella placed underthe control of the promoter of another gene of the same family. Tandemcopies of a hybrid gene consisting of the pertactin structural geneunder the regulation of the FHA promoter, were introduced into the PRNlocus to generate strain 1091-297 which expresses approximately 20 timesthe amount of pertactin as the wild-type 10536 strain.

We have demonstrated that strains of B. pertussis can be engineered toeliminate the production of specific antigens or to overproduce specificantigens as a result of an increase in gene copy number. The additionalgene copies may be single or multiple copies placed at the natural ornovel loci through homologous recombination. They may be native ormutated genes or novel hybrid genes. They also may be heterologous genesfrom other bacterial species. The use of gene deletion and subsequentinsertion of single or multiple natural or novel genes has greatpotential for optimizing the production of antigens for new vaccineformulations. Undesirable genes may be removed, expression of antigensproduced in limiting amounts may be enhanced, and purificationprocedures simplified by the use of the techniques described herein. Therequirement for certain growth supplements may be eliminated as a resultof the efficient expression of selected antigens.

EXAMPLES

Methods of molecular genetics, protein biochemistry, and fermentationand hybridoma technology used but not explicitly described in thisdisclosure and these Examples are amply described in the scientificliterature and are well within the ability of those skilled in the art.

Example 1

This Example illustrates the generic techniques used for vector andstrain constructions and their molecular characterizations.

DNA manipulations, Southern blot analysis, and nick-translation ofprobes were according to Sambrook et al. (Molecular cloning: alaboratory manual/second edition. Cold Spring Harbor Laboratory, ColdSpring Harbor, N.Y. [1989]). Restriction enzymes were used according tothe manufacturers' specifications. Genes were cloned from a B. pertussis10536 genomic library prepared in lambda Charon 35 (described in EPO322,115, U.S. Pat. No. 5,085,862). Plasmid DNA for electroporetictransformation was prepared according to Ish-Horowicz and Burke (Nucl.Acids Res. 9, 2989 [1988]). Chromosomal DNA for Southern blots wasprepared according to Yacoob and Zealey (Nucl. Acids Res. 16, 1639[1988]).

Example 2

This Example describes the procedure for gene replacement in theBordetella pertussis chromosome by unmarked allelic exchange.

The same principle applies to both specific gene deletion and insertionof additional genes. Specific gene deletion, in fact, involves theinsertion of a marker cassette. B. pertussis was transformed byelectroporation as described in Zealey et al., Bio/Technology 8, 1025[1990]. Briefly, cells were grown in 1 liter of Stainer Scholte mediumsupplemented with heptakis(2,6-0-dimethyl)β-cyclodextrin (Imaizumi etal., Infect. Immun. 41, 1138 [1983]) to a density of about 5×10⁹cells/ml and harvested by centrifugation (5000×g, 15 min., 4° C.). Thecells were washed twice with 500 ml of distilled water and once in 50 mlof 10% glycerol, then resuspended in 10 ml of 10% glycerol, aliquotted,and frozen at -70° C. For transformation, 200 ul of cells were combinedwith 10-50 ug of linearized DNA or 5 ug of circular plasmid DNA andincubated on ice for 10 min. The mixture was subjected to a 650 Vexponential decay pulse across a 0.8 mm electrode gap using a BTXTransfector 100 equipped with a Power Plus unit (Biotechnologies andExperimental Research, San Diego Calif.). One ml of medium was added andthe cells incubated with shaking at 36° C. For integration of TOXalleles at the TOX locus of a TOX⁻ strain, cells were transformed withlinearized DNA. After transformation and one hour of non-selectivegrowth, 50 ug/ml of ampicillin was added to the culture, and theincubation continued for 15-24 h at 36° C. One ml samples were platedonto Bordet-Gengou medium containing 100 ug/ml streptomycin andtransformants appeared after 3-5 days of incubation at 36° C.

To increase the success of correct integration at the FHA locus, FHAdeleted B. pertussis strains were transformed in two stages.Electroporation of circular plasmid DNA was performed and integration ofthe entire plasmid was selected for by resistance to ampicillin (50μg/ml) Ap^(R), Tet^(R), Str^(S) transformants were obtained at afrequency of about 500 per ug of DNA, and were presumably due to theintegration of the entire plasmid at either the TOX or FHA locus. Whenthese primary transformants were streaked onto medium containingstreptomycin, Str^(R) colonies were obtained in which the S12/Tet^(R)gene cassette was replaced by the TOX operon by intramolecularrecombination.

Example 3

This Example illustrates the growth of strains and antigencharacterization.

Strains were routinely grown in 10 liter ChemAp bioreactors in modifiedStainer-Scholte medium containing 0.2% heptakis(2,6-O-dimethyl)β-cyclodextrin (Imaizumi et al, Infect. Immun. 41., 1138[1983]) with controls for temperature, pH, and dissolved oxygen, or in10 ml culture. Culture supernatants were tested for antigen productionin antigen-specific ELISAs using monospecific polyclonal and/ormonoclonal antibodies. PT was measured in a fetuin-capture ELISA asdescribed in Loosmore et al. (Infect. Immun. 58, 3653 [1990]). FHA wascaptured by a mouse monoclonal anti-FHA antibody purified from ascitesfluid and the second antibody was a polyclonal rabbit IgG anti-FHAconjugated to horseradish peroxidase. Purified antigens were used asstandards.

Pertussis toxin was purified as described in EPO 322,115, U.S. Pat. No.5,085,862 and U.S. Pat. No. 4,997,915 assigned to the assignee hereofand the disclosure of which is incorporated herein by reference. Acomparative analysis of PT analogues produced from strain 989-56 and thetriple copy TOX* strain 890-393, showed no difference in SDS-PAGEpatterns and HPLC profiles, as shown in FIGS. 4A, 4B and 4C. Table 2below summarizes the biological properties of native PT and PT analogueproduced by wild-type and engineered strains. Results indicate that PTsecreted by single-copy or multiple-copy strains are functionallyequivalent.

Example 4

This Example illustrates the generation of the FHA-deleted strain390-101.

The FHA operon was originally cloned in two steps. An internal 4.5 kbBamH I/BamH I fragment of the FHA B structural gene was cloned from apEV-based expression library (R. Crowl et al., Gene 38, 31 [1985]) usinga polyclonal anti-FHA antibody as probe. This fragment then wasnick-translated and used to probe a Charon phage pertussis genomiclibrary to clone the whole operon.

An 11 kb internal EcoR I/EcoR I fragment of the FHA structural gene wasdeleted and replaced by the S12/Tet^(R) gene cassette to generateplasmid pGZ71, which therefore has 2.5 kb of Bgl II/EcoR I 5'-FHAflanking sequence and 1.7 kb of EcoR I/Cla I 3'-FHA flanking sequence(FIG. 1A).

FIG. 1B demonstrates the construction of plasmid pGZ71. Briefly, plasmidS-2224-1 contains a chromosomal clone representing the 3'-Bgl II/EcoR Iportion of the FHA operon and the S-3595-9-1 plasmid a chromosomal cloneof the 5'-EcoR I/Bgl II portion of the FHA operon. If the two clones arejoined at the Bgl II site, the entire FHA structural gene isreconstituted. Plasmid S-2224-1 was cleaved with Bgl II and EcoR I togive a pBR322-based vector fragment containing 1.7 kb of FHA 3'-flankingregion (3' FHA). Plasmid S-3595-9-1 was digested with Bgl II and EcoR Iand the 2.5 kb fragment containing the FHA 5'-flanking sequence (5'FHA)was ligated with the vector piece from S-2224-1 to yield plasmidS-3616-2. Plasmid pGZ62 contains the S12/Tet^(R) gene cassette in apBR322-based plasmid and is described in detail in EPO 322,115, U.S.Pat. No. 5,085,862. Plasmid S-3616-2 was digested with EcoR I, filled inusing Klenow polymerase, and dephosphorylated with calf alkalinephosphatase (CAP). pGZ62 was digested with EcoR I and Bal I, and filledin. The S-3616-2 vector fragment and pGZ62 insert fragment were ligatedto give pGZ71. The filled in EcoR I and Bal I sites regenerate the EcoRI site.

Electroporetic transformation of a spontaneous streptomycin resistantderivative of B. pertussis strain 10536, i.e. B. pertussis strain str29(ATCC 53972), by linearized pGZ71 led to the deletion of the FHAstructural gene by allelic exchange and yielded the FHA⁻ strain 390-101(ATCC 55157). The deletion of the FHA gene was confirmed by Southernblot analysis and FHA-specific ELISA. Similarly, plasmid pGZ71 was usedto delete the FHA gene from B. pertussis strain 989-56 containing a TOX*operon to generate B. pertussis strain 490-324.

Example 5

This Example describes the generation of the FHA-deleted strain 590-208which contains two copies of TOX at different genetic loci.

A pBR322-based plasmid (S-3741-2) was constructed which contains asingle copy of the 4.7 kb EcoR I/EcoR I TOX operon between the FHAflanking region as described in Example 4. FIG. 1B describes theconstruction of plasmid S-3741-2. Briefly, S-3616-2 was digested withEcoR I, which opened the plasmid between the FHA flanking regions, anddephosphorylated with CAP. Plasmid J-169-1 is a pUC-based clone of TOXwhich was digested with EcoR I to produce an EcoR I/EcoR I TOX fragment.Upon ligation, S-3741-2 was generated which contains a single copy ofthe wild-type TOX operon between the FHA flanking regions.

Introduction of S-3741-2 by electroporation into the FHA-deleted strain390-101, generated a strain having two non-tandem copies of TOX, strain590-208. Southern blot analysis of strain 590-208 genomic DNA showed thecorrect placement of the additional TOX operon at the FHA locus.

When grown in liquid culture, the yield of PT was twice that obtainedfrom the native strain as shown in Tables 1A and 1B below, but therewas, as expected, no FHA production. Plasmid S-3741-2 has been depositedwith the ATCC (accession number 75031).

Example 6

This Example illustrates the generation of the FHA-deleted strain890-393, which contains three copies of the TOX* operon.

A pBR322-based plasmid (S-3822-10) was constructed to harbour two tandemcopies of the 4.7 kb EcoR I/EcoR I TOX* operon between the FHA flankingregions described in Example 4 (FIG. 1A). The construction of S-3822-10is illustrated in FIG. 1C. Briefly, plasmid S-3484-3-15 is a pUC-basedvector containing the TOX 5'- and 3'-flanking sequences surrounding amutated TOX operon (TOX*). The TOX* operon contains the mutationsdesigned to produce a detoxified PT analogue with the Lys9Gly129substitutions in its S1 subunit (Loosmore et al., Infect. Immun. 58,3653 [1990]). S-3616-2 was digested with EcoR I and dephosphorylated.S-3484-3-15 was digested with EcoR I and the TOX* fragment cleaved out.A 5 molar excess of the EcoR I-EcoR I TOX* insert was used in theligation reaction to force in tandem gene copies and plasmid S-3822-10was obtained which contains two adjacent copies of TOX* between the FHAgene flanking regions, as confirmed by restriction enzyme analysis.

Strain 989-56 (ATCC 53975) is the derivative of the 10536 wild-typestrain which expresses the Lys9Gly129 PT analogue (FIG. 2). pGZ71 wasused to delete the FHA structural gene from 989-56 to give strain490-324, which is TOX* FHA⁻. Introduction of S-3822-10 into 490-324 ledto the generation of strain 890-393 which contains three copies of theTOX* operon, two at the FHA locus and one at the original TOX locus(FIG. 2A). When grown in liquid culture, as described in Example 3, theexpression of PT analogue was found to be 3 to 4 times that obtainedfrom strain 10536, as shown in Tables 1A and 1B below. FIG. 3demonstrates the kinetics of PT analogue expression from B. pertussisstrain 890-393.

FIG. 2B shows the Southern blot analysis of genomic DNA from strain890-393. Chromosomal DNA was isolated, restricted simultaneously withBgl II and Cla I and probed with a nick-translated TOX-specific probe.The hybridization pattern of restricted genomic DNA from B. pertussis890-393 (lane 4) was compared with that of DNA obtained from strain10536 (lane 1) and strain 989-56 (lane 2), and revealed the presence ofthe 3.3 and 13 kb TOX-specific fragments characteristic of the nativeTOX operon. For strain 890-393, however, the predicted additionalTOX-specific fragments of 5.3, 5.0, and 3.7 kb were observed. Thehybridization pattern indicated that this strain had one TOX* operon atthe TOX locus and two more copies in tandem at the FHA locus. Strain890-393 has been deposited with the ATCC (accession number 55206).

Example 7

This Example describes the generation and characterization of strain191-134 which contains two tandem copies of TOX* at the TOX locus.

A pUC-based plasmid (S-4000-5) was constructed which contains two tandemcopies of the TOX* operon, between the 5' and 3' TOX flanking regions.The 4.7 kb EcoR I/EcoR I TOX* fragment is defined as the operon and the2.9 kb Sma I/EcoR I 5'-flanking and 4 kb EcoR I/Sal I 3'-flankingregions of the native TOX operon were used to direct the tandem genes tothe TOX locus (see FIG. 1A). The TOX flanking regions have beenextensively described in EPO 322,115, U.S. Pat. No. 5,085,862.

FIG. 1C illustrates the construction of plasmid S-4000-5. Briefly, thepUC-based plasmid S-3484-3-15, which contains the TOX* operon betweenthe TOX flanking regions, was linearized with Kpn I anddephosphorylated. Plasmid S-3822-10 which contains tandem TOX* operonsbetween the FHA flanking regions, was restricted with Kpn I and the 4.6kb Kpn I/Kpn I fragment removed. Upon ligation of these pieces, plasmidS-4000-5 containing two tandem copies of TOX* between the TOX flankingregions was generated.

S-4000-5 was linearized with Hind III and strain 29-8 was transformed byelectroporation to produce strain 191-134. Southern blot analysis of191-134 genomic DNA showed the correct in situ placement of the tandemTOX* operons at the TOX locus, as shown in FIG. 2, lane 3. ChromosomalDNA was obtained from strain 191-134 and digested simultaneously withBgl II and Cla I. When hybridized with a TOX-specific probe, the 13 and3.3 kb bands which indicate the presence of a single copy of TOX at theTOX locus were observed, but an additional 5.0 kb band also wasdetected. This pattern indicates the presence of two tandem copies ofTOX* at the TOX locus. When grown in liquid culture, the kinetics of PTanalogue production by strain 191-134 are similar to those of the nativestrain but 2 to 3 times more PT analogue was produced as shown in Tables1A and 1B below. Strain 191-134 has been deposited with the ATCC(accession number 55205).

Example 8

This Example illustrates the engineering and characterization of strain1190-74 which contains two tandem copies of the wild-type TOX operonintegrated at the TOX locus.

A pUC-based plasmid (S-3980-9) was constructed which is similar toS-4000-5, which contains two tandem copies of TOX* (Example 7), exceptthat the TOX operon is wild-type. S-3980-9 was linearized with Hind IIIand introduced into the TOX deleted strain 29-8 by electroporetictransformation to generate strain 1190-74. Southern blot analysis of1190-74 genomic DNA showed correct in situ placement of the tandemoperons.

When grown in liquid culture, the kinetics of PT production from strain1190-74 was similar to that obtained for the native strain but the yieldof PT was 2 to 3 times higher, as shown in Tables 1A and 1B below.Plasmid S-3980-9 has been deposited with the ATCC (accession number75033).

Example 9

This Example illustrates the effect of PT overproduction on therequirement for cyclodextrin in the medium.

To optimize the expression of PT from a native strain, the medium istypically supplemented with a dextrin derivative, such as 0.2%(heptakis(2,6-O-dimethyl)β-cyclodextrin (Imaizumi et al., Infect. Immun.41, 1138 [1983]). FIG. 5A demonstrates that the addition of cyclodextrinsignificantly enhances the concentration of PT in the fermentation brothof the wild-type strain 10536. Strain 1190-74 contains two tandem copiesof the wild-type TOX operon. When grown in the absence of 0.2%heptakis(2,6-O-dimethyl)β-cyclodextrin, there is a negligible effect onthe expression of PT, as illustrated in FIG. 5B. Similar results wereobserved for the PT analogue-producing strains. In addition, theoverproduction of PT or PT analogue did not affect the expression ofother antigens, such as FHA, agglutinogens, or pertactin.

Example 10

This Example illustrates the generation of the PRN deleted strain1090-108-3.

The PRN gene region was cloned as an 11.3 kb fragment from a Charonphage pertussis library using specific oligonucleotide probes. Theoligonucleotide sequences were AATGAACATGTCTCTGTCACGCATTGTCAA (SEQ. ID:No. 1) and TTCCACGCGGGCTACCGGTACAGCTGGTAA (SEQ. ID: No. 2) andrepresented sequence from base 144 to base 173 and base 2848 to base2877 of the published PRN sequence, respectively (Charles et al., Proc.Natl. Acad. Sci. 86, 3554 [1989]). Plasmids S-3596-1-1 and S-3770-4 arechromosomal subclones of the entire pertactin gene region, PRN (FIG.1D). Digestion of S-3770-4 with Cla I deleted the internal 7 kb ClaI/Cla I fragment of the coding sequence and re-ligation gave plasmidS-3818-1. This pUC-based plasmid contains the 1.6 kb of Sau3A I/Cla I5'-flanking sequence and 3.5 kb of Sau3A I/Cla I 3'-flanking sequencefrom PRN. S-3818-1 was digested with EcoR I and Cla I, filled in usingKlenow polymerase, and dephosphorylated. Plasmid pGZ62 was digested withEcoR I and Bal I to remove the S12/Tet^(R) gene cassette, which then wasfilled in. Ligation of the fragments gave pRY46-10 which contains theselection cassette between the PRN flanking regions (FIG. 1A).

When linearized PRY46-10 was introduced into strain str29 (wild type,ATCC no. 53972), strain 1090-108-3 was generated in which the PRN generegion has been deleted. Southern blot analysis confirmed thereplacement of the PRN gene sequences by the S12/Tet^(R) gene cassette.Pertactin-specific ELISAs demonstrated that strain 1090-108-3 did notproduce this antigen. Strain 1090-108-3 has been deposited with the ATCC(accession number 55156).

Example 11

This Example illustrates the generation of B. pertussis strain 1091-297which contains two tandem copies of the FHApPRN alleles integrated atthe PRN locus.

A pUC-based plasmid (S-4258-7) was constructed that contained two copiesof the FHApPRN hybrid gene sandwiched between the 5'- and 3'-flankingregions of PRN. FIG. 1E illustrates the construction of plasmidS-4258-7. The construction of hybrid genes is described in detail in EPO453,216, U.S. Ser. No. 687,231. Briefly, plasmid S-3595-9-1 is achromosomal subclone of the FHA 5'-flanking region and 5'-portion ofFHAB including the FHA promoter region. Plasmid S-3596-1-1 is achromosomal subclone of the PRN gene locus including the flankingregions. Plasmid S-3595-9-1 was digested with EcoR I and Hinf I togenerate a 240 bp EcoR I/Hinf I fragment containing most of the FHApromoter. Plasmid S-3596-1-1 was digested with EcoR I and Nco I togenerate a 4 kb piece containing pUC with part of the 3'-flanking regionof PRN. A 93 bp Hinf I/Nco I oligonucleotide was used to join thefragments and generate plasmid S-4116-7, which therefore contains thewhole FHA promoter and part of the PRN signal sequence up to the Nco Isite. Plasmid S-3596-1-1 was digested with Nco I to generate a 2.2 kbfragment containing most of the PRN structural gene. Plasmid S-4116-7was linearized with Nco I and dephosphorylated with calf alkalinephosphatase. Ligation of the structural gene fragment behind the FHApromoter gave plasmid S-4131-9. Plasmid S-3770-4 is a chromosomalsubclone of the PRN gene locus including flanking regions. Digestion ofS-3770-4 with Kpn I and EcoR I removes the natural PRN promoter and partof the structural gene. Plasmid S-4131-9 was digested with EcoR I andKpn I and the fragment containing the FHA promoter and 5'-half of thePRN structural gene, was ligated into the S-3770-4 vector fragment togenerate plasmid S-4143-1 which thus has one copy of the FHApPRN hybridgene. S-4143-1 was digested with BamH I and Hind III and the 5.6 kbfragment containing the 5'-PRN and FHApPRN hybrid gene was inserted intopUC digested with BamH I and Hind III to generate plasmid S-4250-1-5which now has the hybrid gene between two EcoR I sites. Plasmid S-4143-1was linearized with EcoR I and dephosphorylated. S-4250-1 was digestedwith EcoR I to release the hybrid gene which was ligated into the EcoR Isite of S-4143-1 to produce plasmid S-4258-7 which has two tandem copiesof the hybrid gene in opposite orientations.

Plasmid S-4258-7 was used to transform the PRN deleted strain 1090-108-3in two stages. Electroporation of circular plasmid DNA was performed andintegration of the entire plasmid selected for by resistance toampicillin and tetracycline. When these primary transformants werestreaked onto medium containing streptomycin, Str^(R) colonies wereobtained in which the S12/Tet^(R) gene cassette was replaced by theFHApPRN hybrid genes by intramolecular recombination. B. pertussisstrain 1091-297 (ATCC 55,462) containing two tandem copies of theFHApPRN hybrid genes was generated in this fashion. Southern blotanalysis showed the correct placement of the two hybrid genes at the PRNlocus as demonstrated in FIG. 6B. When grown in liquid culture, thekinetics of pertactin production were similar to the wild-type strainbut the total yield of protein was approximately 20-fold higher (Table3).

Example 12

This Example describes the construction of B. pertussis strains whichoverproduce both pertussis toxin and pertactin.

In the EPO 453,216 and U.S. Ser. No. 687,231, the inventors describedthe generation of hybrid genes as a means to improve antigen expression.In Example 11 above, the construction of plasmid S-4143-1, whichcontains an FHApPRN hybrid gene, is described. Plasmid S-4143-1 was usedto transform the PRN--deleted B. pertussis strain 1090-108-3 (ATCC55156) and produce strain 591-473 (ATCC 55,321), which contains a singlecopy of the hybrid FHApPRN gene at the PRN locus. The TOX operon of B.pertussis 591-473 was replaced by the selectable S12/Tet^(R) cassette,as described in Example 4 above, to generate B. pertussis strain 891-98.Two tandem copies of wild-type TOX or mutant TOX* were integrated at theTOX locus by transformation with plasmids S-3980-9 or S-4000-5,respectively, as described in Examples 7 and 8 above. This actiongenerated B. pertussis strain 1091-107 (ATCC 55,313) which overexpresseswild-type PT and pertactin and strain 1091-359 (ATCC 55,312) whichoverexpresses the Lys9Gly129 PT analogue and pertactin. FIGS. 7A and 7Billustrate the levels of antigen production from strains 1091-107 and1091-359 when grown in 10 liter fermentors.

Table 1 provides details of the production of PT and FHA from certainstrains of Bordetella pertussis wherein modification to the TOX operonhas been effected. The effect of the presence of multiple gene copies onantigen production can be seen from the data presented (see Examples 7and 8).

Table 2 provides details of the biological characterization of certainstrains of B. pertussis.

Table 3 provides details of the production of PT and FHA from certainstrains of B. pertussis wherein modification to the PRN operon has beeneffected. The effect of the presence of multiple hybrid gene copies onantigen production can be seen from the data provided (see Example 11).

                                      TABLE 1A                                    __________________________________________________________________________    Production of pertussis toxin (PT) and filamentous haemagglutinin (FHA)       by strains                                                                    of Bordetella pertussis that contain multiple copies of the TOX operon.       Strains contained                                                             either the wild-type TOX operon or the genetically detoxified Lys9 Gly129     allele located at                                                             either the TOX or FHA loci as indicated. They were grown in 10 ml             shake-flasks and antigens                                                     in culture supernatants determined by antigen-specific ELISAs.                                                Antigen Production                                Gene at TOX                                                                           Gene at FHA                                                                           Transformed (μg/ml).sup.a                              Strain                                                                            Locus   Locus   Strain Plasmid                                                                            PT    FHA                                     __________________________________________________________________________    10536                                                                             TOX.sup.wild-type                                                                     FHA                 4.7 ± 0.6                                                                        58.7 ± 4.5                           29-8                                                                              Δ FHA                 0     48.1 ± 3.4                           390-101                                                                           TOX.sup.wild-type                                                                     Δ Str29  pGZ71                                                                              4.1 ± 0.9                                                                        0                                       490-324                                                                           TOX.sup.Lys9Gly129                                                                    Δ 989-56 pGZ71                                                                              3.9 ± 0.8                                                                        0                                       590-208                                                                           TOX.sup.wild-type                                                                     TOX.sup.wild-type                                                                     390-101                                                                              S-3741-2                                                                           9.4 ± 0.8                                                                        0                                       1190-74                                                                           2TOX.sup.wild-type                                                                    FHA     29-8   S-3980-9                                                                           20.6 ±  1.5                                                                      54.2 ± 2.4                           191-134                                                                           2TOX.sup.Lys9Gly129                                                                   FHA     29-8   S-4000-5                                                                           12.9 ± 1.0                                                                       49.3 ± 3.88                          890-393                                                                           TOX.sup.Lys9Gly129                                                                    2TOX.sup.Lys9Gly129                                                                   490-324                                                                              S-3822-10                                                                          19.9 ± 1.2                                                                       0                                       __________________________________________________________________________     .sup.a Expressed as mean ± standard errors at 95% confidence limits fo     at least four independent experiments.                                        Δ deleted gene.                                                    

                                      TABLE 1B                                    __________________________________________________________________________    Production of pertussis toxin (PT) and filamentous haemagglutinin (FHA)       by strains                                                                    of Bordetella pertussis that contain multiple copies of the TOX operon.       Strains contained                                                             either the wild-type TOX operon or the genetically detoxified Lys9 Gly129     allele                                                                        located at either the TOX or FHA loci as indicated. They were grown in 10     bioreactors and antigens in culture supernatants determined by                antigen-specific ELISAs.                                                                                      Antigen                                       Gene at     Gene at Transformed Production (mg/l)                             Strain                                                                            TOX Locus                                                                             FHA Locus                                                                             Strain Plasmid                                                                            PT  FHA                                       __________________________________________________________________________    10536                                                                             TOX.sup.wild-type                                                                     FHA                 20  90                                        989-56                                                                            TOX.sup.Lys9Gly129                                                                    FHA                 20  80                                        590-208                                                                           TOX.sup.wild-type                                                                     TOX.sup.wild-type                                                                     390-101                                                                              S-3741-2                                                                           40  0                                         1190-74                                                                           2TOX.sup.wild-type                                                                    FHA     29-8   S-3980-9                                                                           52  73                                        191-134                                                                           2TOX.sup.Lys9Gly129                                                                   FHA     29-8   S-4000-5                                                                           34  70                                        890-393                                                                           TOX.sup.Lys9Gly129                                                                    2TOX.sup.Lys9Gly129                                                                   490-324                                                                              S-3822-10                                                                          80  0                                         __________________________________________________________________________

                                      TABLE 2                                     __________________________________________________________________________    Biological characterization of PT analogues obtained from Bordetella          pertussis                                                                     strains that over-produce pertussis toxin.                                                    Relative CHO                                                      Number of   Cell    Relative                                                                             Histamine                                                                            Mouse                                       TOX   PT    Clustering                                                                            ADPR   Sensitization                                                                        Protection                              Strain                                                                            Operons                                                                             Produced                                                                            Activity (%)                                                                          Activity (%)                                                                         (LD.sub.50 μg).sup.a                                                              (ED.sub.50 μg).sup.b                 __________________________________________________________________________    10536                                                                             1     Wild-type                                                                           100     100    0.05   --                                      989-56                                                                            1     Lys.sup.9 Gly.sup.129                                                               ≦0.0005                                                                        <0.0001                                                                              >50.sup.c                                                                            4                                       890-393                                                                           3     Lys.sup.9 Gly.sup.129                                                               ≦0.0005                                                                        <0.0001                                                                              >50.sup.c                                                                            4                                       __________________________________________________________________________     .sup.a The LD.sub.50 is the dose required to kill 50% of the mice             following challenge with histamine acid phosphate (1 mg/10 g body weight)     .sup.b The ED.sub.50 is the dose which protects 50% of the mice from an       intracerebral challenge with B. pertussis 18323. This value cannot be         determined for wildtype PT because of the toxicity of the molecule.           .sup.c No toxicity at the indicated dose.                                

                  TABLE 3                                                         ______________________________________                                        Production of pertussis toxin (PT) , filamentous hemagglutinin                (FHA) and pertactin, by Bordetella pertussis strains that                     contain one or two copies of the hybrid PRN allele at                         the PRN locus. The strains were grown in 10 ml shake-flasks                   and antigen levels in culture                                                 supernantants determined by antigen-specific ELISAs.                                       Antigen Expression (μg ml.sup.-1).sup.a                       Strain  Construction                                                                             PT       FHA     Pertactin                                 ______________________________________                                        10536   wild-type  5.9 ± 1.1                                                                           53.7 ± 10.1                                                                        8.9 ± 3.6                              1090-108-3                                                                            PRN Δ                                                                              1.6 ± 0.9                                                                           31.9 ± 9.8                                                                         0                                         591-473 FHApPRN    4.7 ± 1.0                                                                           47.9 ± 9.4                                                                         73.6 ± 12.1                            1091-297                                                                              2xFHApPRN  4.1 ± 2.0                                                                           86.7 ± 10.1                                                                        186.7 ± 15.8                           ______________________________________                                         .sup.a Concentrations are expressed as means ± standard errors at 95%      confidence limits for at least five independent experiments.                  Δ: deleted gene.                                                   

SUMMARY OF DISCLOSURE

In summary of this disclosure, the present invention provides novelstrains of Bordetella which provide altered levels of gene expression,permitting enhanced antigen production and optimum purification.Modifications are possible within the scope of this invention.

    __________________________________________________________________________    SEQUENCE LISTING                                                              (1) GENERAL INFORMATION:                                                      (iii) NUMBER OF SEQUENCES: 2                                                  (2) INFORMATION FOR SEQ ID NO:1:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 30 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                                       AATGAAC ATGTCTCTGTCACGCATTGTCAA30                                             (2) INFORMATION FOR SEQ ID NO:2:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 30 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                                       TTCCA CGCGGGCTACCGGTACAGCTGGTAA30                                         

What we claim is:
 1. A genetically-modified Bordetella strain from whichthe FHA gene is absent from the genome of the strain and which containsan additional TOX gene in the genome of the strain.
 2. The Bordetellastrain of claim 1 which is identified as Bordetella pertussis strain590-208 and which is derived from FHA⁻ Bordetella pertussis strain390-101 having ATCC accession number 55157 by chromosomal transformationemploying plasmid S-3741-2 having ATCC accession number
 75031. 3. Agenetically-modified Bordetella strain from which the FHA gene is absentfrom the genome of the strain and which contains agenetically-detoxified mutant TOX* gene in the genome of the strain. 4.The Bordetella strain of claim 3 wherein said genetically-detoxifiedmutant TOX* gene encodes a pertussis toxin analog having mutationsGlu129→Gly129 and Arg9→Lys9 in the S1 subunit of pertussis toxin.
 5. TheBordetella strain of claim 3 which contains two tandemly-linkedgenetically-detoxified TOX* genes at the FHA locus and a single copy ofthe genetically-detoxified TOX* gene at the TOX locus and which isidentified as Bordetella pertussis strain 890-393 having ATCC accessionnumber
 55206. 6. A genetically-modified Bordetella strain containing atleast two tandem copies of a TOX gene or of a genetically-detoxifiedmutation thereof in the genome of the strain.
 7. The Bordetella strainof claim 6 containing two TOX* genes encoding a pertussis toxin analoghaving the mutations Arg9→Lys9 and Glu129→Gly129 in the S1 subunit ofpertussis toxin and which is identified as Bordetella pertussis strain191-134 having ATCC accession number
 55205. 8. The Bordetella strain ofclaim 6 containing two TOX (wild-type) genes and which is identified asBordetella pertussis strain 1190-74 which is derived from the TOX⁻Bordetella pertussis strain 29-8 having ATCC accession number 53973 bychromosomal transformation employing plasmid S-3980-9 having ATCCaccession number
 75033. 9. A genetically-modified Bordetella straincontaining, in the genome thereof, at least two copies (tandem ornon-tandem) of a hybrid gene comprising the PRN structural gene underthe regulation of the FHA promoter.
 10. The Bordetella strain of claim 9containing two tandem FHApPRN genes and identified as Bordetellapertussis strain 1091-297.
 11. A genetically-detoxified Bordetellastrain containing at least two copies of the TOX (wild-type) gene in thegenome of the strain in combination with one or more hybrid genes in thegenome of the strain, said hybrid gene comprising a Bordetella geneunder the regulation of a promoter of another Bordetella gene.
 12. TheBordetella strain of claim 11 which is identified as Bordetellapertussis strain 1091-107 having ATCC accession number
 55313. 13. Agenetically-modified Bordetella strain containing at least two copies(tandem or non-tandem) of a genetically-detoxified TOX* gene in thegenome of the strain in combination with one or more hybrid genes in thegenome of the strain, said hybrid gene comprising a Bordetella geneunder the regulation of a promoter of another Bordetella gene.
 14. TheBordetella strain of claim 13 wherein said TOX* gene encodes a pertussistoxin analogue having the mutations Arg9→Lys9 and Glu129→Gly129 in theS1 subunit of pertussis toxin.
 15. The Bordetella strain of claim 14which is identified as Bordetella pertussis strain 1091-359, having ATCCaccession number
 55312. 16. Plasmid S-3741-2 having ATCC accessionnumber
 75031. 17. Plasmid S-3980-9 having ATCC accession number 75033.18. A vaccine against Bordetella infection comprising, as a singlecomponent or as one component of a multicomponent vaccine, a killedBordetella strain of claims 1, 3, 6, 9, 11 or
 13. 19. A process ofproducing an antigen, which comprises:culturing a genetically-modifiedBordetella strain of claims 1, 3, 6, 9, 11 and 13 in a culture medium toeffect expression of a protein encoded by genes present in said strain.20. The process of claim 19 wherein said protein is extracted from saidculture medium and detoxified for vaccine use.